The invention relates to processes for magnetorelaxometric qualitative and/or quantitative detection of analytes in liquid and solid phases, compounds for magnetorelaxometric detection, and their use in analysis and immunomagnetography.
It is known that immunoscintigraphy makes it possible to detect pathological structures in vivo with the aid of radiolabeled structure-specific substances, which are also referred to below as markers. To this end, antibodies that are labeled with .gamma.-rays or antibody fragments are usually used. In addition, other structure-specific substances, such as, e.g., peptides or oligonucleic or polynucleic acids are also used or are being researched. The portion of specifically bound radioactivity is, however, generally small in all these processes. Consequently, in the case of these studies, the level of markers that are not specifically bound and thus circulate in the blood or accumulate in organs such as the liver, kidney, efferent urinary passages, or bladder is very high. In many cases, this high background radiation impedes adequate detection of pathological structures. Panchapakesan [Immunol. Cell Biol., 70 (1992) 295] and Ziegler [New England Journal of Medicine, 324 (1991) 430] therefore refer to ways of improving immunoscintigraphy. Such ways are also described in EP 0 251 494. The goal of most of the processes is to accelerate the elimination of radioactivity that is not specifically bound.
In addition, the use of antibodies that are conjugated with paramagnetic or superparamagnetic substances or antibody fragments for locating pathological structures in vivo has been proposed on various occasions. To date, nuclear spin tomography or magnetometry that is based on changes in susceptibility (WO 93/05818 and WO 91/15243) have been considered as detection processes for such labeled antibodies. In the case of these detection processes, the problem of the variable portion of the signal owing to unbound portions of the marker as well as owing to natural variations in the susceptibility and relaxivity of the tissue also remains present. In addition, the methods often are not sensitive enough to be able to detect just small amounts of specifically bound markers.
A process that makes it possible to detect only the portion of bound markers and thus is not influenced by the extent of the unbound markers is not known, however.
It is also known that quantitative immunoassays as well as other binding assays (e.g., receptor binding assays) make it possible to determine a very large number of substances that can also be of biological relevance in samples of varying composition. Generally, however, only one parameter per sample in an assay is determined in this way. An existing survey of the various processes is: T. Chard [An Introduction to Radioimmunoassay and Related Techniques: Laboratory Techniques in Biochemistry and Molecular Biology, 4th ed., Elsevier Science Publishers, Amsterdam (1990)]. The basis of all binding assays is the high detection sensitivity of compounds that are labeled with isotopes or by some other means with the high specificity of ligand-receptor reactions.
The known assay processes have the following drawbacks, however:
The processes for simultaneous determination of various analytes within the same sample are based on the binding of various radio-, fluorescence- or enzymologically-labeled probes to the analytes. In this case, the unbound or bound activity of the probes for quantitative determination of the analyte is generally measured after subsequent separation and washing. In this case, the amount of usable different probe labels is greatly limited. Thus, for example, in the case of different radioisotopes as probe labels, so-called overlapping phenomena occur which lead to a rapid loss of the quantitative accuracy of individual signals. The combination of various enzymes as probe labels causes comparable problems, whereby the feasibility here is further hampered by the necessary search for reaction conditions that allow the simultaneous determination of enzyme reactions in a system. PA1 The sensitivity of the process is limited by, for example, non-specific interactions between matrix and probe, or else by limited labeling capability on the part of the probe (low specific activity). PA1 The successful implementation of the process often requires that the sample material obtained be worked up (e.g., production of serum or plasma from whole blood, extraction of samples with organic solvents, concentration of the analyte using chromatographic processes, etc.). PA1 For successful implementation of the processes, separation and washing steps, which are used in the separation of bound and unbound receptors or ligands, are essential in most cases. PA1 To carry out radioimmunoassays, the use of radiating nuclides, which are costly and complicated to handle, is necessary. PA1 In practice, the storage of previously used markers often causes problems since they are either unstable (radioimmunoassays) and must therefore constantly be made up fresh or else react in a sensitive manner to environmental influences. PA1 i) Turning of the whole colloidal particle inside the surrounding liquid, whereby the time constant depends on the hydrodynamic diameter of the particles including the shell, the viscosity of the carrier liquid, and temperature, which mainly reflects parameters of the environs of the particles; this mechanism is also referred to below as Brownian relaxation or extrinsic superparamagnetism, PA1 ii) Turning of the internal magnetizing vector inside the colloidal particles, whereby the time constant depends in a very sensitive manner on material and shape (the anisotropy constants of the particle material used), volume and the temperature of the particles used. These are basically intrinsic parameters of the particles; this mechanism is also referred to below as Neelian relaxation or intrinsic superparamagnetism. PA1 i) are labeled with ferrimagnetic or ferromagnetic colloidal particles and then PA1 ii) these magnetically labeled analytes are used in a liquid or immobilized sample that is to be measured, the substances that specifically bind the analytes are added, and the sample that is to be measured is magnetized with the aid of a magnetic field that is applied from the outside and, after the outside field is turned off, the relaxation of the magnetization of the magnetic markers is measured with the aid of magnetic field sensors. PA1 i) being labeled with the ferrimagnetic or ferromagnetic colloidal particles that relax in the time range of the measurement, whereby the ferrimagnetic or ferromagnetic colloidal particles are selected in such a way that under the measurement conditions, the Brownian relaxation has a shorter relaxation time than the Neelian relaxation and then PA1 ii) these magnetically labeled substances being used in an immobilized sample that is to be measured, and the sample that is to be measured being magnetized with the aid of a magnetic field of suitable intensity that is applied from the outside and, after the outside field is turned off, the relaxation of the magnetization of the magnetic markers being measured with the aid of magnetic field sensors, whereby the different relaxation behaviors of solid-phase-bound and unbound magnetic markers are used for analysis. As a measurement variable, the complex susceptibility of the samples can also be determined as a function of frequency. PA1 i) being labeled with ferrimagnetic or ferromagnetic colloidal particles, whereby the ferrimagnetic or ferromagnetic colloidal particles are selected in such a way that under the measurement conditions the Brownian relaxation has a shorter relaxation time than the Neelian relaxation and then PA1 ii) these magnetically labeled substances being used in a sample that is to be measured, and the sample that is to be measured being magnetized with the aid of a magnetic field of suitable intensity that is applied from outside and, after the outside field is turned off, the relaxation of the magnetization of the magnetic markers being measured with the aid of magnetic field sensors, whereby the different relaxation behaviors of the magnetic markers bound with the analyte relative to the unbound magnetic markers are used for analysis. PA1 1. Production of as homogeneous a magnetic field as possible in advantageous volume, turning off the field and measuring the spatial distribution of the relaxing magnetic field with the aid of a multichannel sensor. Said sensor should enclose the measurement object as completely as possible. For the production of sufficient measurement information, repeated measurement with sequential rastering of the measurement object is also possible. PA1 2. Sequential production of a local field that is limited in space, turning off the field and measuring the spatial distribution of the relaxing magnetic field with the aid of a single-channel sensor. The use of a multichannel sensor is also possible.